1. Field of the Invention
This invention relates to methods for procuring decellularizing and further processing and dry preserving collagen-based tissues derived from humans and animals for transplantation into humans or other animals. These methods produce a tissue product that consists of a selectively preserved extracellular protein matrix that is devoid of certain viable cells which normally express major histocompatibility complex antigenic determinants and other antigens which would be recognized as foreign by the recipient. This extracellular protein matrix is made up of collagen and other proteins and provides a structural template which may be repopulated with new viable cells that would not be rejected by the host. These viable cells may be derived from the host (autologous cells) before or after transplantation or from an alternative human source including foreskin, umbilical cord or aborted fetal tissues. More particularly, this invention relates to the procurement and processing of collagen-based tissues such that complications following implantation (including but not limited to immunorejection, contracture, calcification, occlusion, and infection) are significantly reduced relative to current implant procedures and materials.
2. Description of the Related Art
Tissue and organ transplantation is a rapidly growing therapeutic field as a result of improvements in surgical procedures, advancements in immunosuppressive drugs and increased knowledge of graft/host interaction. Despite major advancements in this field, modern tissue transplantation remains associated with complications including inflammation, degradation, scarring, contracture, calcification (hardening), occlusion and rejection. There are numerous investigations underway directed toward the engineering of improved transplantable tissue grafts, however, it is generally believed in the industry that ideal implants have yet to be produced.
Autologous or self-derived human tissue is often used for transplant procedures. These procedures include coronary and peripheral vascular bypass surgeries, where a blood vessel, usually a vein, is harvested from some other area of the body and transplanted to correct obstructed blood flow through one or more critical arteries. Another application of autologous tissue is in the treatment of third degree burns and other full-thickness skin injury. This treatment involves grafting of healthy skin from uninjured body sites to the site of the wound, a process called split-skin grafting. Additional applications of autologous tissue transplantation include bone, cartilage and fascia grafting, used for reconstructive procedures.
The motive for using autologous tissue for transplantation is based upon the concept that complications of immunorejection will be eliminated, resulting in enhanced conditions for graft survival. Unfortunately, however, other complications can ensue with autologous transplants. For example, significant damage can occur to several tissue components of transplanted veins during harvesting and prior to implantation. This damage can include mechanical contraction of the smooth muscle cells in the vein wall leading to loss of endothelium and smooth muscle cell hypoxia and death. Hypoxic damage can result in the release of cellular lysosomes, enzymes which can cause significant damage to the extracellular matrix. Following implantation, such damage can lead to increased platelet adhesion, leucocyte and macrophage infiltration and subsequently further damage to the vessel wall. The end result of such damage is thrombosis and occlusion in the early post implant period. Even in the absence of such damage, transplanted autologous veins typically undergo thickening of the vessel wall and advancing atherosclerosis leading to late occlusion. The exact cause of this phenomena is uncertain but may relate to compliance mismatch of the vein in an arterial position of high blood pressure and flow rate. This phenomena may be augmented and accelerated by any initial smooth muscle cell and matrix damage occurring during procurement. Occlusion of transplanted veins can necessitate repeat bypass procedures, with subsequent re-harvesting of additional autologous veins, or replacement with synthetic conduits or non-autologous vessels.
Another example of complications resulting from autologous tissue transplantation is the scarring and contracture that can occur with split-skin grafts for full-thickness wound repair. Split-skin grafts are typically mechanically expanded by the use of a meshing instrument, which introduces a pattern of small slits in the skin. The split-skin graft is then stretched to cover a larger wound area. Dividing epidermal cells will ultimately grow into and cover the areas of the slits, however, the underlying dermal support matrix does not readily expand into these areas. The dermal matrix, composed primarily of collagen, other extracellular protein matrix proteins, and basement membrane complex, is responsible for the tensile, flexible nature of skin. Absence of a dermal matrix results in scarring and contracture in the area of the slits. This contracture can be severe and in cases of massively burned patients that undergo extensive split-skin grafting, can necessitate subsequent release surgical procedures to restore joint movement.
When the supply of transplantable autologous tissues is depleted, or when there is no suitable autologous tissue available for transplant (e.g., heart valve replacement), then substitutes may be used, including man-made synthetic materials, animal-derived tissues and tissue products, or allogeneic human tissues donated from another individual (usually derived from cadavers). Man-made implant materials include synthetic polymers (e.g. (PTFE) polytetrafluroethylene, Dacron and Goretex) sometimes formed into a tubular shape and used as a blood flow conduit for some peripheral arterial bypass procedures. Additionally, man-made synthetics (polyurethanes) and hydrocolloids or gels may be used as temporary wound dressings prior to split-skin grafting.
Other man-made materials include plastics and carbonized metals, fashioned into a prosthetic heart valve, utilized for aortic heart valve replacement procedures. Synthetic materials can be made with low immunogenicity but are subject to other limitations. In the case of mechanical heart valves, their hemodynamic characteristics necessitate life-long anticoagulant therapy. Synthetic vascular conduits, often used in above-the-knee peripheral vascular bypass procedures, are subjected to an even higher incidence of occlusion than autologous grafts. In many cases, a preference is made for a biological implant which can be a processed animal tissue or a fresh or cryopreserved allogeneic human tissue.
Animal tissues (bovine or porcine) chemically treated are commonly used as replacements for defective human heart valves, and have been used in the past for vascular conduits. The concept in the chemical processing is to stabilize the structural protein and collagen matrix by cross-linking with glutaraldehyde or a similar cross-linking agent. This treatment also masks the antigenic determinants, such that the human host will not recognize the implant as foreign and precludes an immunorejection response. Glutaraldehyde-treated tissues, however, will not allow in-migration of host cells which are necessary for remodeling, and will gradually harden as a result of calcification. For this reason, glutaraldehyde-treated tissues generally require replacement in 5-7 years. Glutaraldehyde-treated bovine veins have been used in the past for vascular bypass bypass procedures, however, their use has been discontinued due to the unacceptable incidence of aneurysm formation and occlusion.
The use of allogeneic transplant tissues has been applied to heart valve replacement procedures, arterial bypass procedures, bone, cartilage, and ligament replacement procedures and to full-thickness wound treatment as a temporary dressing. The allogeneic tissue is used fresh, or may be cryopreserved with the use of DMSO and/or glycerol, to maintain viability of cellular components. It is thought that the cellular components contain histocompatibility antigens, and are capable of eliciting an immune response from the host. In many cases, the patient receiving the allogeneic transplant undergoes immunosuppressive therapy. Despite this therapy, many allogeneic transplants, including heart valves and blood vessels, undergo an inflammatory response, and fail within 5-10 years. Allogeneic skin is typically rejected within 1-5 weeks of application, and has never been demonstrated to be permanently accepted by the host, even with the use of immunosuppressive drugs.
Alternative processing methods have been developed by others that are intended to address the limitations of allogeneic and animal-derived transplant tissues. Freeze-drying is used routinely in the processing of allogeneic bone for transplantation. It has been found that the freeze drying process results in a graft which elicits no significant rejection response as compared to fresh or cryopreserved allogeneic bone. The freeze-dried bone following implant acts as a template, which is subsequently remodelled by the host. When the freeze-drying process has been applied to more complex tissues such as heart valves, the results have been mixed but overall unsatisfactory. A study was conducted in which 15 allogeneic heart valves were processed by freeze-drying prior to transplantation. Most of the freeze-dried valves failed due to mechanical causes in the early post-graft interval. Those freeze-dried valves which did not fail, however, demonstrated prolonged functionality (up to 15 years).
Enzymes and detergent processing has also been used to remove antigenic cells from collagen-based transplantable tissues. Organic solvents and detergent treatments have been used successfully with relatively simple tissues such as dura mater used in reconstructive surgical procedures. Chemical processing of more complex structures such as heart valves, vascular conduits and skin, however, has had only limited success in clinical applications.
The invention of this patent is a comprehensive processing technique that addresses potential damaging events in the preparation of complex collagen-based tissues for transplantation. The technology combines both biochemical and physical processing steps to achieve the ideal features of template function such that the tissue graft can be remodeled for long-term maintenance by the host.